PROJECT SUMMARY For pathogens, the ability to acquire iron is critical and one of the best-understood indicators of virulence. Numerous pathogenic organisms take advantage of the abundance of heme in the host cell and the human diet as a source of essential iron. Until recently however, all of the known heme degrading enzymes required molecular oxygen for function. In this project, we will investigate the mechanism of anaerobic heme degradation in enterohemorrhagic E. coli (EHEC), a facultative anaerobic pathogen (e.g. O157:H7, which causes bloody diarrhea, hemolytic uremic syndrome, kidney failure and death). We recently found that the ChuW enzyme from EHEC catabolyzes heme and liberates iron under strictly anaerobic conditions. This newly identified anaerobic heme degradation enzyme is part of an important module of heme utilization proteins (ChuW, X, Y in E. coli) that is also found in other aggressive pathogens such as Vibrio cholerae. Identification and characterization of this new pathway in enteric pathogens provides an unexplored opportunity for developing novel antimicrobial compounds. In addition, the enzyme at the heart of this investigation (ChuW) is a radical SAM methyltransferase (RSMT); it utilizes a [4Fe-4S] cluster to generate a powerful radical species that facilitates the liberation of iron from heme through a methyl transfer reaction and chemical rearrangement of the porphyrin. ChuW is capable of methylating an otherwise unreactive sp2- hybridized carbon atom. Furthermore, ChuW is a member of the class C RSMTs, which are poorly understood but have already been shown to catalyze key reactions in the biosynthesis of novel compounds with antitumor and antibiotic properties. This work will provide new insight into this important class of RSMTs and how they control highly reactive radicals to facilitate specific chemical conversions in the biosynthesis of compounds including anti-microbial and anti-tumor agents. Using a multifaceted approach that combines traditional enzyme kinetics, rigorous spectroscopic techniques, and modern structural biology tools, we will characterize the mechanism of anaerobic heme degradation by ChuW, the properties of the anaerobic catabolites, and the interplay of two additional proteins with ChuW in the anaerobic degradation of heme as well as the transport and further reduction of the catabolites. The latter is important given recent work showing that heme degradation products play a vital role in regulation of heme flux and iron homeostasis in other aggressive pathogens. Our specific aims are to determine whether: ChuW catalyzes the liberation of iron from heme via a radical SAM methyltransferase mechanism (using 5'-dAdo? and resulting in formation of ?anaerobilin?) (Aim 1); ChuX facilitates the chelation of the iron atom and delivery of ?anaerobilin? to ChuY (Aim 3); and ChuY catalyzes the NADPH-dependent reduction of anaerobilin to ?anaerorubin? (Aim 2). Our long term goal is to provide the necessary mechanistic insight to selectivity target the anaerobic heme degradation pathway, which appears to be specifically associated with a select set of aggressive, opportunistic, and pathogenic bacteria.